A number of methods exist for introducing exogenous genetic material to cells, which methods have been used for a wide variety of applications including, for example, research uses to study gene function, and ex vivo or in vivo genetic modification for therapeutic purposes. Ex vivo genetic modification involves the removal of specific cells from an animal, including humans, introduction of the exogenous genetic material, and then re-introduction of the genetically modified cells into the animal. By contrast, in vivo genetic modification involves the introduction of genetic material directly to the animal, including humans, using an appropriate delivery vehicle, where it is taken up by the target cells.
Generally, the various methods used to introduce nucleic acids into cells have as a goal the efficient uptake and expression of foreign genes. In particular, the delivery of exogenous nucleic acids in humans and/or various commercially important animals will ultimately permit the prevention, amelioration and cure of many important diseases and the development of animals with commercially important characteristics. The exogenous genetic material, either DNA or RNA, may provide a functional gene which, when expressed, produces a protein lacking in the cell or produced in insufficient amounts, or may provide an antisense DNA or RNA or ribozyme to interfere with a cellular function in, e.g., a virus-infected cell or a cancer cell, thereby providing an effective therapeutic for a disease state.
Engineered viruses are commonly used to deliver genes to cells. Viral vectors are generally efficient in gene delivery but have certain drawbacks, for example stimulation of an immune response when delivered in vivo. As a result, therefore, a number of non-viral nucleic acid delivery systems have been and continue to be developed. Thus, for example, cationic lipids are commonly used for mediating nucleic acid delivery to cells. See, for example, U.S. Pat. No. 5,264,618, which describes techniques for using lipid carriers, including the preparation of liposomes and pharmaceutical compositions and the use of such compositions in clinical situations. Other non-viral gene delivery systems likewise involve positively-charged carrier molecules, for example, peptides such as poly-L-lysine, polyhistidine, polyarginine, or synthetic polymers such as polyethylimine and polyvinylpyrrolidone.
Nucleic acids are generally large polyanionic molecules which, therefore, bind cationic lipids and other positively-charged carriers through charge interactions. It is believed that the positively charged carriers (or polycations), form tight complexes with the nucleic acid, thereby condensing it and protecting it from nuclease degradation. In addition, polycationic carriers may act to mediate transfection by improving association with negatively-charged cellular membranes by giving the complexes a positive charge, and/or enhancing transport from the cytoplasm to the nucleus where DNA may be transcribed.
For cationic lipid-mediated delivery, the cationic lipids typically are mixed with a non-cationic lipid, usually a neutral lipid, and allowed to form stable liposomes, which liposomes are then mixed with the nucleic acid to be delivered. The liposomes may be large unilamellar vesicles (LUVs), multilamellar vesicles (MLVs) or small unilamellar vesicles (SUVs). The liposomes are mixed with nucleic acid in solution, at concentrations and ratios optimized for the target cells to be transfected, to form cationic lipid-nucleic acid transfection complexes. Alterations in the lipid formulation allow preferential delivery of nucleic acids to particular tissues in vivo. See PCT patent application numbers WO 96/40962 and WO 96/40963.
With respect to any of the polycationic nucleic acid carriers, transfection efficiency is highly dependent on the characteristics of the polycation/nucleic acid complex. The nature of the complex that yields optimal transfection efficiency depends upon the mode of delivery, e.g. ex vivo or in vivo; for in vivo delivery, the route of administration, e.g., intravenous, intramuscular, intraperitoneal, inhalation, etc.; the target cell type, etc. Depending on the use, therefore, different carriers will be preferred. In addition to the choice of polycationic carrier, transfection efficiency will depend on certain physical characteristics of the complexes as well, such as charge and size. These characteristics depend largely on the method by which the complexes are prepared. Particularly for human therapeutic purposes, therefore, it is desirable to have a method of forming the nucleic acid/polycationic carrier complexes in a highly controllable manner. Further, it is desirable to have a process for preparing the complexes which is highly reproducible and scaleable.
The present invention provides these and related advantages as well.
Relevant Literature
Cationic lipid carriers have been shown to mediate intracellular delivery of plasmid DNA (Felgner et al., (1987) Proc. Natl. Acad. Sci. (USA), 84:7413-7416); mRNA (Malone et al., (1989) Proc. Natl. Acad. Sci. (USA) 86:6077-6081); and purified transcription factors (Debs et al., (1990) J. Biol. Chem. 265: 10189-10192), in functional form. Literature describing the use of lipids as carriers for DNA include the following: Zhu et al., (1993) Science, 261:209-211; Vigneron et al., (1996) Proc. Natl. Acad. Sci. USA, 93:9682-9686; Hofland et al., (1996) Proc. Natl. Acad. Sci. USA, 93:7305-7309; Alton et al., (1993) Nat. Genet. 5:135-142; von der Leyen et al., (1995) Proc. Natl. Acad. Sci. (USA), 92:1137-1141; See also Stribling et al., (1992) Proc. Natl. Acad. Sci (USA) 89:11277-11281, which reports the use of lipids as carriers for aerosol gene delivery to the lungs of mice. For a review of liposomes in gene therapy, see Lasic and Templeton, (1996) Adv. Drug Deliv. Rev. 20:221-266.
The role of helper lipids in cationic lipid-mediated gene delivery is described in Felgner et al., (1994) J. Biol. Chem. 269(4): 2550-2561 (describing improved transfection using DOPE); and Hui et al., (1996) Biophys. J. 71: 590-599. The effect of cholesterol on liposomes in vivo is described in Semple et al., (1996) Biochem. 35(8): 2521-2525.
The use of cationic peptides and proteins in DNA delivery is described in Emi et al., (1997) Biochem Biophys Res. Comm. 231(2):421-424 (polyarginine); Fritz et al., (1996) Hum. Gene Ther. 7(12):13951404 (histone H1 and SV40 large T antigen nuclear localizing signal); Gao and Huang (1996) Biochemistry 35(3) 1027-1036 (poly-L-lysine, protamine); Legendre and Szoka (1993) Proc. Natl. Acad. Sci USA 90(3):893-897 (gramicidin S); and Niidome et al., (1997) J. Biol. Chem. 272(24):15307-15312 (cationic alpha-helical oligopeptides). Additional transfection facilitating agents are described in Ibanez, et al., (1996) Biochem Cell Biol 74(5):633-643 (spermidine); Budker et al., (1997) Biotechniques 23(1):139 (histone H1 and amphipathic polyamines); and Barthel et al., (1993) DNA Cell Biol. 12(6):553-560 (lipospermine).
A method of preparing cationic lipid/nucleic acid transfection complexes by first forming lipid micelles in the presence of detergent is described in WO 96/37194. Methods of preparing DNA-lipid complexes using polyethylene glycol-phospholipid conjugates and polyamines are described in Hong et al., (1997) 400(2):233-237.